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Biomedical subjects

A B Borle

Publications and source records attributed to A B Borle.

At least 37 records · Page 2Linked to original sources
PubMed1992

Fructose protects rat hepatocytes from anoxic injury. Effect on intracellular ATP, Ca2+i, Mg2+i, Na+i, and pHi.

The effects of fructose on the intracellular ionic changes evoked by anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with sodium-binding benzofuran isophthalate, intracellular pH (pHi) with 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, and viability by trypan blue exclusion. ATP, Pi, phosphomonoesters, and the cell phosphorylation potential assessed by the reciprocal of the Pi/ATP ratio were measured by 31P NMR spectroscopy in real time. Intracellular free Mg2+ (Mg2+i) was calculated from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. When the perfusate contained 5 mM glucose as substrate, anoxia caused a fall in ATP, a rise in Pi, and in the Pi/ATP ratio, a biphasic increase in Ca2+i that reached 1.45 +/- 0.42 microM and a 6-fold increase in LDH. When 15 mM fructose was used as substrate during the anoxic period, intracellular ATP decreased much faster than with glucose, Pi did not increase, and the concentration of phosphomonoesters increased 2.5-fold. During the first hour of anoxia, the Pi/ATP ratio was higher in the fructose than in the glucose group indicating that the hepatocyte phosphorylation potential and ATP decreased faster and to lower levels with fructose than with glucose. On the other hand, ATP and the phosphorylation potential of the fructose group increased during the second hour of anoxia, in contrast to their continuous decline in the glucose group. The major surge in Ca2+i was depressed 52% when glucose was replaced by fructose: Ca2+i reached only 0.7 +/- 0.2 microM instead of 1.45 +/- 0.42 microM (p less than 0.01). Anoxia also caused an increase in Na+i and an intracellular acidosis. The rise in Na+i was significantly greater with fructose than with glucose. Na+i rose from a control value of 15.9 +/- 2.4 to 32.2 +/- 0.4 mM with glucose and to 48.7 +/- 0.7 mM with fructose (p less than 0.001). The decrease in pHi from a control value of 7.43 +/- 0.03 was consistently greater and faster with fructose than with glucose: 6.59 +/- 0.03 and 7.04 +/- 0.01, respectively. At the same time, fructose completely suppressed LDH release and reduced the loss of viability produced by anoxia from 27.7 +/- 2.9 to 14 +/- 3.1% (p less than 0.05).

Adenosine Triphosphate↗
PubMed1992

Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.

The effects of anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with SBFI, intracellular pH (pHi) with BCECF, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, ATP by 31P NMR spectroscopy in real time, and intracellular free Mg2+ (Mg2+i) from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. After 1 h of anoxia, beta-ATP fell 66%, and 85% after 2 h, while the Pi/ATP ratio increased 10-fold from 2.75 to 28.3. Under control conditions, the resting cytosolic free calcium was 127 +/- 6 nM. Anoxia increased Ca2+i in two distinct phases: a first rise occurred within 15 min and reached a mean value of 389 +/- 35 nM (p less than 0.001). A second peak reached a maximum value of 1.45 +/- 0.12 microM (p less than 0.001) after 1 h. During the first hour of anoxia, Na+i increased from 15.9 +/- 2.4 mM to 32.2 +/- 1.2 mM (p less than 0.001), Mg2+i doubled from 0.51 +/- 0.05 to 1.12 +/- 0.01 mM (p less than 0.001), and pHi decreased from 7.41 +/- 0.03 to 7.06 +/- 0.1 (p less than 0.001). LDH release doubled during the first hour and increased 6-fold during the second hour of anoxia. Upon reoxygenation, ATP, Ca2+i, Mg2+i, Na+i, and LDH returned near the control levels within 45 min. To determine whether the increased LDH release was related to the rise in Ca2+i, and whether the increased Ca2+i was caused by Ca2+ influx, the cells were perfused with Ca(2+)-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca(2+)-free medium, beta-ATP again fell 90%, but Ca2+i, after the first initial peak, fell below control levels, and LDH release increased only 2.7-fold. During reoxygenation, Ca2+i, ATP, Na+i, and LDH returned near the control levels within 45 min. These results suggest that the rise in Ca2+i induced by anoxia is caused by an influx of Ca2+ from the extracellular fluid, and that LDH release and cell injury may be related to the resulting rise in Ca2+i.

Adenosine Triphosphate↗
PubMed1992

Na(+)-Ca2+ antiporter activity of rat hepatocytes. Effect of adrenalectomy on Ca2+ uptake and release from plasma membrane vesicles.

The presence and mode of Na(+)-Ca2+ antiporter activity were studied in hepatocytes isolated from sham-operated or adrenalectomized rats and in inside-out plasma membrane vesicles isolated from rat liver. Decreasing extracellular Na+ (Na+o) immediately increased cytosolic free calcium (Ca2+i). The rise in Ca2+i was proportional to the reduction in Na+o and was caused by an increased calcium influx, presumably on the Na(+)-Ca2+ antiporter operating in the reverse mode. Perfusing the cells with Ca(2+)-free media stimulated Ca2+ efflux and decreased Ca2+i, an effect dependent on Na+o. This suggests an activation of the forward mode of Na(+)-Ca2+ exchange. There was little difference in these parameters between sham and adx groups. In contrast, steady-state calcium uptake by inside-out plasma membrane vesicles was inhibited 40% after adrenalectomy. The decreased calcium uptake was not caused by a deficiency in the ATP-dependent Ca2+ pump, whose Km and Vmax were unaffected by adrenalectomy, but by an Na(+)-dependent leak from the vesicles. Ca2+ efflux was proportional to the extravesicular Na+ concentration, suggesting that the calcium leak may take place on a Na(+)-Ca2+ antiporter. This Na(+)-dependent calcium efflux was significantly increased in vesicles prepared from adx rat livers. These results suggest that hepatocytes have functional Na(+)-Ca2+ antiporters that can operate in both forward and reverse modes. Under normal conditions, the Na(+)-Ca2+ antiporter apparently operates in the reverse mode as a Ca2+ influx pathway. The increase in Na(+)-dependent Ca2+ efflux evoked by adrenalectomy in plasma membrane vesicles could explain the recent results we obtained in hepatocytes isolated from adx rats, showing increased calcium influx, increased Ca2+i, increased intracellular calcium sequestration, and increased plasmalemmal calcium cycling.

Adrenalectomy↗
PubMed1991

Effects of pH on Ca2+i, Na+i, and pHi of MDCK cells: Na(+)-Ca2+ and Na(+)-H+ antiporter interactions.

The effects of high and low extracellular pH (8.0 and 6.8) on the intracellular concentration of Ca2+, Na+, and H+ were measured in perfused Madin-Darby canine kidney (MDCK) cells cast in agarose gel threads. Cytosolic free Ca2+ (Ca2+i) was measured with aequorin, while intracellular Na+ (Na+i) and H+ (Hi+ or pHi) were determined with the fluorescent indicators SBFI and BCECF, respectively. In addition, H+ secretion was assayed by the pH-stat method, and Na+ or Ca2+ fluxes were measured with 22Na or 45Ca, respectively. H+ secretion was significantly depressed by several experimental conditions that are known to inhibit the Na(+)-H+ antiporter: H+ secretion decreased 44% in the presence of 10(-5) M ethylisopropylamiloride, 49% in Na+o-free media, 44% in the presence of 10(-4) M ouabain, and 32% in the presence of 10(-4) M 8-bromoadenosine 3',5'-cyclic monophosphate. In addition, pHi decreased by 0.2 pH units in Na+o-free media. Finally, recovery from an intracellular acidosis evoked by 20 mM NH4Cl pulse required the presence of extracellular Na+. When the extracellular pH (pHo) was increased from 7.4 to 8.0, H+ secretion increased 58% from 17.5 to 27.7 nmol.min-1.mg protein-1 and Na+ influx increased 48%. As a result, pHi rose from 7.43 to 7.71 and Na+i increased from 15.6 to 19.7 mM. Finally, Ca2+i rose from 120 to 268 nM. These results suggest that the high pHo stimulated the Na(+)-H+ antiporter, and the subsequent rise in Na+i decreased the Na+ electrochemical potential, thereby activating the reverse mode of Na(+)-Ca2+ exchange (Ca2+ influx vs. Na+ efflux) which led to the rise in Ca2+i.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Chloride↗
PubMed1990

Effect of adrenalectomy on Ca2+ signaling in rat hepatocytes.

To pursue our studies of the effects of adrenalectomy on the adrenergic regulation of phosphorylase a, cAMP, cell calcium, and Ca2+ signaling in rat hepatocytes (Studer, R.K., and Borle, A.B. (1984) Biochim. Biophys. Acta 804, 377-385; Freudenrich, C.C., and Borle, A.B. (1988) J. Biol. Chem. 263, 8604-8610), we have further examined the alpha 1-adrenergic pathway in adrenalectomized and sham-operated male rats. We measured the number and affinity of alpha 1-adrenergic receptors, the cytosolic free Ca2+ concentration [(Ca2+]i) of hepatocytes with aequorin, inositol triphosphate (IP3) accumulation, and Ca2+ influx and efflux across the plasma membrane. We also compared the effects of vasopressin with those obtained with epinephrine. We found that the number of alpha 1-adrenergic receptors was slightly depressed (-23%), but that their affinity was unchanged. However, IP3 accumulation evoked by epinephrine was decreased 50%. This is probably the main cause for the depressed peak rise in [Ca2+]i we previously observed and reported. We also found that the basal resting Ca2+ influx was increased after adrenalectomy. Experiments with the beta-blocker propranolol, which abolished the epinephrine-evoked increase in Ca2+ influx, suggest that this effect may be mediated by cAMP, at least in adrenalectomized animals. The effects of vasopressin on IP3 [Ca2+]i and Ca2+ influx and efflux were also significantly decreased after adrenalectomy, indicating that alpha 1-adrenergic-mediated and other IP3-dependent Ca2+ signaling pathways are depressed after adrenalectomy.

Adrenalectomy↗
PubMed1990

Effects of low extracellular Ca2+ on cytosolic free Ca2+, Na+, and pH of MDCK cells.

The effects of lowering the Ca2+ electrochemical potential (delta mu Ca2+) by removing extracellular calcium (Ca2+o) was studied in Madin-Darby canine kidney (MDCK) cells. We measured cytosolic free calcium (Ca2+i), intracellular Na+ (Na+i), and intracellular pH (pHi) as well as Ca2+ and Na+ influx and efflux and H+ secretion. As soon as Ca2+o was lowered to zero, Ca2+ efflux increased 2-fold, and Na+ influx increased 4.3-fold. As a result, Ca2+i fell 71% from 120 to 34.7 nM, and Na+i rose 56% from 16.7 to 26.1 mM. At the same time, H+ secretion was depressed 38%, and pHi fell from 7.32 to 7.22. When Ca2+o was restored to normal (1.3 mM), there was a sharp rise in Ca2+i from 34.7 to 243 nM (+600%) caused by an immediate increase in Ca2+ influx (+112%). At the same time, Na+ efflux increased 20%. Ten to fifteen minutes after restoring Ca2+o to 1.3 mM, Ca2+i, Na+i, and pHi were back to control levels. These results suggest that reducing delta mu Ca2+ activates the forward mode of Na(+)-Ca2+ exchange which leads to a fall in Ca2+i and a decreased delta mu Na+ by increasing Na+i. The fall in delta mu Na+ secondarily depresses Na(+)-H+ exchange which results in a reduced H+ secretion and a fall in pHi. When Ca2+o is restored to 1.3 mM, the reverse mode of Na(+)-Ca2+ exchange is activated, since delta mu Ca2+ is larger than normal because of the low Ca2+i, and delta mu Na+ is reduced because of the high Na+i.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

An overview of techniques for the measurement of calcium distribution, calcium fluxes, and cytosolic free calcium in mammalian cells.

An array of techniques can be used to study cell calcium metabolism that comprises several calcium compartments and many types of transport systems such as ion channels, ATP-dependent pumps, and antiporters. The measurement of total cell calcium brings little information of value since 60 to 80% of total cell calcium is actually bound to the extracellular glycocalyx. Cell fractionation and differential centrifugation have been used to study intracellular Ca2+ compartmentalization, but the methods suffer from the possibility of Ca2+ loss or redistribution among cell fractions. Steady-state kinetic analyses of 45Ca uptake or desaturation curves have been used to study the distribution of Ca2+ among various kinetic pools in living cells and their rate of Ca2+ exchange, but the analyses are constrained by many limitations. Nonsteady-state tracer studies can provide information about rapid changes in calcium influx or efflux in and out of the cell. Zero-time kinetics of 45Ca uptake can detect instantaneous changes in calcium influx, while 45Ca fractional efflux ratio, can detect rapid stimulations or inhibitions of calcium efflux out of cells. Permeabilized cells have been successfully used to gauge the relative role of intracellular organelles in controlling [Ca2+]i. The measurement of the cytosolic ionized calcium ([Ca2+]i) is undoubtedly the most important and, physiologically, the most relevant method available. The choice of the appropriate calcium indicator, fluorescent, bioluminescent, metallochromic, or Ca2(+)-sensitive microelectrodes depends on the cell type and the magnitude and time constant of the event under study. Each probe has specific assets and drawbacks. The study of plasma membrane vesicles derived from baso-lateral or apical plasmalemma can also bring important information on the (Ca2(+)-Mg2+) ATPase-dependent calcium pump and on the kinetics and stoichiometry of the Na(+)-Ca2+ antiporter. The best strategy to study cell calcium metabolism is to use several different methods that focus on a specific problem from widely different angles.

Animals↗
PubMed1988

The effects of adrenalectomy on the alpha-adrenergic regulation of cytosolic free calcium in hepatocytes.

We have previously published that bilateral adrenalectomy in the rat reduces the Ca2+-mediated alpha-adrenergic activation of hepatic glycogenolysis, while it increases the cellular calcium content of hepatocytes. In the experiments presented here, the concentration of cytosolic free calcium (Ca2+i) at rest and in response to epinephrine was measured in aequorin-loaded hepatocytes isolated from sham and adrenalectomized male rats. We found that in adrenalectomized rats the resting Ca2+i was elevated, the rise in Ca2+i evoked by epinephrine was reduced, and the rise in 45Ca efflux that follows such stimulation was depressed. Furthermore, the slope of the relationship between Ca2+i and calcium efflux was decreased 60% in adrenalectomized. Adrenalectomy did not change Ca2+ release from intracellular calcium pools in response to IP3 in saponin-permeabilized hepatocytes. The EC50 for inositol 1,4,5-triphosphate and the maximal Ca2+ released were similar in both sham and adrenalectomized animals. Finally, the liver calmodulin content determined by radioimmunoassay was not significantly different between sham and adrenalectomized rats. These results suggest that 1) adrenalectomy reduces calcium efflux from the hepatocyte, probably by an effect on the plasma membrane (Ca2+-Mg2+)-ATPase-dependent Ca2+ pump and thus alters cellular calcium homeostasis; 2) adrenalectomy decreases the rise in Ca2+i in response to epinephrine; 3) this decreased rise in Ca2+i is not due to defects in the intracellular Ca2+ storage and mobilization processes; and 4) the effects of adrenalectomy on cellular calcium metabolism and on alpha-adrenergic activation of glycogenolysis are not caused by a reduction in soluble calmodulin.

Adrenalectomy↗
PubMed1987

Intracellular messenger for action of angiotensin II on fluid transport in rabbit proximal tubule.

These studies were designed to examine the cellular messenger that mediates the action of angiotensin II on fluid transport (Jv) in the rabbit proximal tubule. We measured the effects of angiotensin II on Jv, activation of adenylate cyclase, and the concentration of cytosolic free calcium (Cai) in the rabbit proximal tubule. In nine rabbit proximal convoluted tubules (PCT), angiotensin II, 10(-8) M and 10(-6) M, decreased Jv by 18 and 25%, P less than 0.05. In eleven rabbit proximal straight tubules (PST), 10(-8) and 10(-6) M angiotensin II decreased Jv by 20 and 23%, P less than 0.02. Angiotensin II did not affect lumen-to-bath phosphate fluxes in PCT or PST, and it did not activate adenylate cyclase in PST. In a preparation of proximal tubules (PCT and PST) loaded with aequorin, angiotensin II, 10(-8) and 10(-6) M, transiently increased Cai by 13 and 32%, P less than 0.001. We propose that Cai surges, activated by angiotensin II, are part of a cellular message that inhibits Jv in the rabbit proximal tubule.

Adenylyl Cyclases↗
PubMed1986

Implications of a rise in cytosolic free calcium in the activation of RAW-264 macrophages for tumor cell killing.

The concentration of cytosolic-free calcium (Ca2+i) was determined with aequorin in RAW-264 macrophage-like cells activated in vitro for tumor cell killing with lymphokine (LK) and lipopolysaccharide (LPS). Treatments of these cells with optimal doses of stimulants, which evoked the development of cytolytic activity, also induced a rise in their Ca2+i. No rise in Ca2+i could be observed under treatments which failed to activate cells. The presence of both stimulants was an absolute requirement for evoking cytolytic activity and also a rise in Ca2+i. There was an apparent parallelism between the rate of activation and the rate of rise in Ca2+i. Cells which slowly developed their cytolytic activity exhibited a slow rise in Ca2+i, while macrophages which acquired their cytolytic activity at the faster rate also showed a more rapid increase in Ca2+i. The development of cytolytic activity in RAW-264 macrophages was inhibited by two intracellular calcium antagonists, TMB-8 and ruthenium red. This inhibition could be reversed by high concentrations of extracellular calcium. TMB-8, at the concentrations which were effective in inhibiting the activation process, also completely blocked the associated rise in Ca2+i. These results suggested that Ca2+i might play a role in the mechanism of tumoricidal transformation of RAW-264 macrophages.

Aequorin↗
PubMed1986

The effect of calmodulin antagonists on the activation of RAW-264 macrophage-like cells for tumor cell killing.

The effects of several calmodulin antagonists on the activation of RAW-264 macrophage-like cells for tumor cell killing were investigated. At concentrations ranging from 5 X 10(-8) to 5 X 10(-7) M, calmidazolium, trifluoperazine, chlorprothixene, chlorpromazine and W-13 inhibited the development of cytolytic activity, evoked in RAW-264 by treatment with lymphokine and lipopolysaccharide, in a dose-dependent manner. Since the order of the potency of these drugs against the activation of RAW-264 cells was much the same as their ability to inhibit calmodulin-dependent phosphodiestherase activity: calmidazolium greater than trifluoperazine greater than chlorprothixene greater than chlorpromazine greater than W-13, and because W-12, a nonactive analog of W-13, failed to inhibit the process of activation, we believe that the development of cytolytic activity in RAW-264 cells may be dependent on calmodulin. At micromolar concentrations, calmodulin antagonists (except calmidazolium) enhanced the process of activation. The enhancement of cytolytic activity was neither the result of the toxicity of these drugs nor related to their effects on intracellular calcium. It was entirely dependent on the presence of stimulants but occurred independently from the stage of macrophage activation, and most likely was due to the nonspecific interference of these agents with calmodulin-independent processes.

Animals↗